Generation of a biologically active, secreted form of human thyroid peroxidase by site-directed mutagenesis.
Using site-directed mutagenesis, we introduced two stop codons immediately upstream of the putative transmembrane domain in human thyroid peroxidase (hTPO) cDNA, truncating the carboxyl terminus of hTPO (933 amino acids) by 85 residues. Mutated hTPO cDNA, inserted into a eukaryotic expression vector, was stably transfected into Chinese hamster ovary (CHO) cells. Immunoprecipitation of cellular 35S-methionine-labeled proteins with Hashimoto's serum revealed a 105-101 kilodalton doublet. In contrast, cells transfected with wild-type hTPO yielded a 112-105 kilodalton doublet. In pulse-chase experiments, CHO cells expressing the truncated hTPO protein secreted immunoprecipitable TPO into the culture medium after 4 h of chase, with levels accumulating progressively over a 24-h period. In contrast, CHO cells expressing wild-type hTPO released no immunoprecipitable TPO into the culture medium. The secreted, truncated form of hTPO appeared as a single band of lesser electrophoretic mobility, as opposed to the doublet expressed within cells. TPO enzymatic activity was present in conditioned media from CHO cells transfected with the mutated hTPO, but was absent in media from cells expressing wild-type hTPO. The stability of the mutated protein appeared similar to that of wild-type hTPO. In summary, we have generated a mutated, secreted form of hTPO that is enzymatically active and immunologically intact. Our data confirm the existence of a transmembrane domain in hTPO, and that hTPO is predominantly an enzyme with an extracellular orientation. The secreted form of hTPO has the potential for generating large amounts of soluble TPO protein for use in future structural and immunological studies.
Foti
D
D
Thyroid Molecular Biology Unit, V.A. Medical Center, San Francisco, California 94121.
Kaufman
K D
KD
Chazenbalk
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GD
Rapoport
B
B
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D006801:Humans; D007453:Iodide Peroxidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D014162:Transfection
D004247:DNA; D007453:Iodide Peroxidase